1. Field of the Invention
The present invention relates to a DNA microchip for identifying fungal species and a method thereof, in particular to a DNA microchip for identification of Phellinus noxius and allied species and a method thereof.
2. The Prior Arts
It has been known that there are about more than 80,000 species of fungi, approximately 8000 species of which are plant pathogens that cause various types of plant diseases in agriculture, horticulture and forestry. Some plant diseases even lead to famine and massive mortality of the forests. The issue of how to rapidly, precisely and sensitively identify fungal pathogens in order to take proper and effective measures for prevention and management in advance, reduce loss of life and property and promote social welfare is indeed of great urgency.
Phellinus noxius, a species of Phellinus, is particularly a notorious fungal pathogen that is considered as forests killer. The fungus infects and colonizes the roots and stem base of the forests and results in tissue degradation and decay and finally the whole plant is dried out. This has been extensively reported in Asia, Africa, Australia and New Zealand. It has been also reported a couple of decades, and particularly prevalent more recently in Taiwan, that at least 140 kinds of forests, including ornamental trees, fruit trees and forestation tree species, have been infected by the fungus, which leads to acute or chronic wilting or drying out, landscape destruction, economic loss and environmental safety. In addition, the other species of Phellinus, for example Phellinus pini and Phellinus werii belonging to the same genus but different species or A. mellea from the genus Armillariella or A. ostoyae from the genus Armillaria, were also reported in the USA, Canada, Australia, New Zealand and Europe to infect massive coniferous forests or broad-leaved forests, which causes poor growth, weakness, early defoliation, advanced blooming, decay, withering and top-over of the plants in windbreak and hence becomes an enormous restricted factor and a potential severe threat to forest management.
Traditionally, the diagnosis of plant diseases is carried out according to Koch's postulates, the process of which includes i) isolation and identification of pathogens from the lesion of the infected plants; ii) multiplication of the pathogens in vitro or in vivo; iii) back inoculation of the pathogens to healthy plants to generate the same disease with identical symptoms; iv) re-isolation and verification of the pathogens. This is a process of re-establishing and verifying the disease cycle. Accordingly, it is obvious that pathogenic identification is the main step in the diagnosis of plant diseases. The diagnosis in tradition principally depends on the features of the microscopic or macroscopic morphology, structure and sporogenesis of a pathogen. However, if anyone wants to correctly identify such diverse and structurally complicated fungi, besides familiarity with distinctions among various forms, to accept learning, education and training, also requires practical operation and experience accumulation over a long period of time and therefore a seriously burden. The dramatic progress in molecular biology in recent years, there is some significant progress, particularly with respect to systematic, phylogeny and identification of plant pathogens, for examples amplified DNA fragment length polymorphism (AFLP), rapid amplification of DNA polymorphism (RAPD), simple sequence repeat (SSR), selected characterized amplified region of DNA (SCAR), nano-magnetic bead polymerase chain reaction (NMB-PCR), multi-plex PCR and suspension microsphere-based array. However, there was no method available for a rapid identification of Phellinus noxiusu and closely related fungal pathogens until 2009, which do severely harm to the forests, so that domestic and foreign quarantine and inspection institutions are not capable of monitoring and rapidly and precisely identifying the fungal pathogens to curtail their accidental introduction. Furthermore, the early symptoms caused by the pathogens often vary with the habitat, growth and age of the plants and the infectious potential of the pathogens. Even the early symptoms are not so remarkable and therefore this makes an accurate diagnosis more difficult. If a rapid diagnosis and a precise identification can be performed in the early phase, this will become the essential factor for prevention of the occurrence and prevalence of such diseases in nursery, or tree species for forestation and selection of habitats. Consequently, the development of a rapid and precise technique for diagnosis and identification is becoming an urgent theme.